The nuclear hormone receptors which include the retinoic acid receptor (RAR) represent a super family of proteins that activate transcription in eukaryots upon binding small bioavailable ligands (i.e. hormones). Recent structural and biochemical studies have revealed much of the molecular details with which RAR and closely related proteins single- handedly recognize and bind their ligands, bind target DNA sequences and activate transcription. This study aims to explore the structural mechanisms with which ligand binding and transactivation functions are coupled using a novel "block and recover" strategy. Based on principles of molecular recognition involving electrostatics and sterics, we will generate site-directed mutants of the ligand binding domain of RAR that block ligand binding and transcriptional activation by the natural ligand, retinoic acid. We will then design and synthesize new ligands that can be used to selectively bind and recover transcriptional activation from these mutant RARs as measured by in vivo transcription assays. Using precedented methods involving site-directed mutagenesis and/or in vivo selections we will also modify the DNA binding domain of RAR to recognize novel DNA sequences. The combined effect of using molecular biology and organic synthesis to change both the ligand binding and DNA binding specificities of RAR is to reconstruct a complete, new ligand-dependent transcription pathway with a unique ligand, receptor and promoter. This would provide a powerful tool that could be flexibly adapted to regulate gene expression in a variety of systems for the study of gene function, cell-cycle regulation and the potential regulation of future gene therapies. By comparing transactivation function to direct measurements of ligand binding using spectroscopic methods in vitro, we will explore which structural modifications affect ligand binding and which affect the protein's intrinsic ability to transactivate. We will compare our "artificial" mutations in RAR, to similar natured mutations found in RAR and in the highly homologous thyroid hormone receptor (TR) that have been implicated in genetic disease. The design strategies developed in the study of RAR will be used to design thyroid hormone analogs that may be capable of restoring activity to mutant TRs that have been found in patients with the genetic disease GRTH (generalized resistance to thyroid hormone). If successful this would represent a new and powerful strategy of treating a genetic disease by rational molecular design.